Process and composition for preparing a lignocellulose-based product, and the product obtained by the process

ABSTRACT

A process for the manufacture of a lignocellulose product, the process comprising the step of mixing in a reaction medium (i) a phenolic polymer being substituted with a phenolic hydroxy group; (ii) a lignocellulose containing material having immobilized to a cellulosic fraction thereof a fusion polypeptide, the fusion polypeptide including an enzyme being capable of catalyzing the oxidation of phenolic groups and a cellulose binding peptide; and (iii) an oxidizing agent. A composition of matter for use in the process and a lignocellulose product obtainable by the process are also disclosed.

RELATED PATENT APPLICATIONS

The present application is a Continuation of U.S. patent applicationSer. No. 10/129,366, filed on May 3, 2002, which is a National Phase ofPCT Application No. PCT/IL00/00665, filed on Oct. 19, 2000, which claimsthe benefit under §119(e) of U.S. Provisional Patent Application No.60/164,140, filed on Nov. 8, 1999, and U.S. Provisional PatentApplication No. 60/166,389, filed on Nov. 18, 1999.

FIELD AND BACKGROUND OF THE INVENTION

The present invention provides a process and compositions for producinga lignocellulose-based product, e.g., fiber board, such as hardboard ormedium-density fiber board (“MDF”), particle board, plywood, paper orpaperboard (such as cardboard and linerboard), from an appropriatelignocellulosic starting material, such as wood fiber or vegetablefiber, having an enzyme adhered thereto via a cellulose binding peptide,which enzyme is capable of catalyzing the oxidation of phenolic groupsof a phenolic polymer which may form an integral part of thelignocellulosic starting material, e.g., lignin, in the presence of anoxidizing agent and optionally in the presence of additionallignocellulosic starting material devoid of the enzyme, e.g., recycledfibers.

The use of the process of the invention confers improved mechanicalproperties on lignocellulose-based products prepared thereby, especiallypaper products such as liner board, cardboard and corrugated board.

Lignocellulose-based products prepared from lignocellulosic startingmaterials, notably products manufactured starting from vegetable fiberor wood fiber prepared by mechanical or mechanical/chemical procedures(the latter often being denoted “semi-chemical” procedures), or by achemical procedure without bleaching, or from wood particles (wood“chips”, flakes and the like), are indispensable everyday materials.

Some of the most familiar types of such products include paper forwriting or printing, cardboard, corrugated cardboard, fiber board (e.g.“hardboard”), and particle board.

Virtually all grades of paper, cardboard and the like are produced fromaqueous pulp slurry. Typically, the pulp is suspended in water, mixedwith various additives and then passed to equipment in which the paper,cardboard etc. is formed, pressed and dried. Irrespective of whethermechanically produced pulp (hereafter denoted “mechanical pulp”),semi-chemically produced pulp (hereafter denoted “semi-chemical pulp”),unbleached chemical pulp or pulp made from recycled fibers (i.e., pulpprepared from recycled fibers, rags and the like) is employed, it isoften necessary to add various strengthening agents to the pulp in orderto obtain an end product having adequate mechanical properties.

In the case of paper and board for use in packaging and the like, thetensile strength and tear strength under dry and wet conditions are ofprimary importance; moreover, notably in the case of certain grades ofcardboard (e.g., so-called unbleached board for the manufacture ofcorrugated cardboard boxes for packaging, transport and the like), thecompression strength of the material is often also an important factor.Among the strengthening agents used today there are a number ofenvironmentally undesirable substances which it would be desirable toreplace by more environmentally acceptable materials. As examples hereofmay be mentioned epichlorohydrin, urea-formaldehyde andmelamine-formaldehyde.

In the case of “traditional” lignocellulose-based composites for use inbuilding construction, flooring, cladding, furniture, packaging and thelike, such as hardboard (which is normally made from wood fibersproduced by mechanical or semi-chemical means or by so-called “steamexplosion”) and particle board (which is made from relatively coarsewood particles, fragments or “chips”), binding of the wood fibers orparticles to give a coherent mass exhibiting satisfactory strengthproperties can be achieved using a process in which the fibers/particlesare treated—optionally in a mixture with one or more “extenders”, suchas lignosulfonates and/or kraft lignin—with synthetic adhesives(typically adhesives of the urea-formaldehyde, phenol-formaldehyde orisocyanate type) and then pressed into the desired form (boards, sheets,panels etc.) with the application of heat.

The use of synthetic adhesives of the above-mentioned types in theproduction of wood products is, however, generally undesirable from anenvironmental and/or safety point of view, since many such adhesives aredirectly toxic—and therefore require special handling precautions—and/orcan at a later stage give rise to release of toxic and/orenvironmentally harmful substances; thus, for example, the release offormaldehyde from certain cured formaldehyde-based adhesives (used asbinders in, e.g., particle board and the like) has been demonstrated.

In the light of the drawbacks associated with the use of syntheticadhesives as binders in the manufacture of lignocellulose-basedproducts, considerable effort has been devoted in recent years to thedevelopment of binder systems and binding processes which are moreacceptable from an environmental and toxicity point of view, andrelevant patent literature in this respect includes the following:

EP 0 433 258 A1 discloses a procedure for the production of mechanicalpulp from a fibrous product using a chemical and/or enzymatic treatmentin which a “binding agent” is linked with the lignin in the fibrousproduct via the formation of radicals on the lignin part of the fibrousproduct. This document mentions “hydrocarbonates”, such as cationicstarch, and/or proteins as examples of suitable binding agents. Asexamples of suitable enzymes are mentioned laccase, lignin peroxidaseand manganese peroxidase, and as examples of suitable chemical agentsare mentioned hydrogen peroxide with ferro ions, chlorine dioxide,ozone, and mixtures thereof.

EP 0 565 109 A1 discloses a method for achieving binding of mechanicallyproduced wood fragments via activation of the lignin in the middlelamella of the wood cells by incubation with phenol-oxidizing enzymes.The use of a separate binder is thus avoided by this method.

U.S. Pat. No. 4,432,921 describes a process for producing a binder forwood products from a phenolic compound having phenolic groups, and theprocess in question involves treating the phenolic compound with enzymesto activate and oxidatively polymerize the phenolic compound, therebyconverting it into the binder. The only phenolic compounds which arespecifically mentioned in this document, or employed in the workingexamples given therein, are lignin sulfonates, and a main purpose of theinvention described in U.S. Pat. No. 4,432,921 is the economicexploitation of so-called “sulfite spent liquor”, which is a liquidwaste product produced in large quantities through the operation of thewidely-used sulfite process for the production of chemical pulp, andwhich contains lignin sulfonates.

With respect to the use of lignin sulfonates—in particular in the formof sulfite spent liquor—as phenolic polymers in systems/processes forbinding wood products (as described in U.S. Pat. No. 4,432,921), thefollowing comments are appropriate: (i) subsequent work (see H. H. Nimzin Wood Adhesives, Chemistry and Technology, Marcel Dekker, New York andBasel 1983, pp. 247-288), and A Haars et al. in Adhesives from RenewableResources, ACS Symposium Series 385, American Chemical Society 1989, pp.126-134) has demonstrated that by comparison with the amounts of“traditional” synthetic adhesives which are required in the manufactureof wood-based boards, very large amounts of lignin sulfonates arerequired in order to achieve comparable strength properties; (ii) thepressing time required when pressing wood-based board products preparedusing lignin sulfonate binders has been found to be very long, see E.Roffael and B. Dix, Holz als Roh-und Werkstoff 49 (1991) 199-205; (iii)lignin sulfonates available on a commercial scale are generally veryimpure and of very variable quality, see J. L. Philippou, Journal ofWood Chemistry and Technology 1(2) (1981) 199-227; (iv) the very darkcolor of spent sulfite liquor renders it unsuited as a source of ligninsulfonates for the production of, e.g., paper products (such aspackaging paper, linerboard or unbleached board for cardboard boxes andthe like) having acceptable color properties.

U.S. Pat. No. 5,846,788, from which the above background information isderived, and which is incorporated by reference as if fully set forthherein, teaches that binding of lignocellulosic materials (vegetablefibers, wood chips, etc.) using a combination of a polysaccharide havingat least substituents containing a phenolic hydroxy group (in thefollowing often simply denoted a “phenolic polysaccharide”), anoxidizing agent and an enzyme capable of catalyzing the oxidation ofphenolic groups by the oxidizing agent can be employed in themanufacture of lignocellulose-based products exhibiting strengthproperties at least comparable to, and often significantly better than,those achievable using previously known processes which have attemptedto reduce or avoid the use of toxic and/or otherwise harmful substances,such as the processes described in EP 0 433 258 A1, EP 0 565 109 A1 andU.S. Pat. No. 4,432,921. Thus, for example, the amount of binderrequired to prepare lignocellulose-based products of very satisfactorystrength by the process described in U.S. Pat. No. 5,846,788 isgenerally much lower typically by a factor of about three or more—thanthe level of binder (based on lignin sulfonate) required to obtaincomparable strength properties using the process according to U.S. Pat.No. 4,432,921. The process according to U.S. Pat. No. 5,846,788 can thusnot only provide an environmentally attractive alternative to moretraditional binding processes employing synthetic adhesives, but it canprobably also compete economically with such processes.

However, the process described in U.S. Pat. No. 5,846,788, requires theuse of purified enzymes which are expensive materials as is compared toother raw materials and reagents used in the process of manufacturinglignocellulose-based products.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a process for producing a lignocellulose-basedproduct, e.g. fiber board, such as hardboard or medium-density fiberboard (“MDF”), particle board, plywood, paper or paperboard (such ascardboard and linerboard), from an appropriate lignocellulosic startingmaterial devoid of the above limitation.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided aprocess for the manufacture of a lignocellulose product, the processcomprising the step of mixing in a reaction medium (i) a phenolicpolymer being substituted with a phenolic hydroxy group; (ii) alignocellulose containing material having immobilized to a cellulosicfraction thereof a fusion polypeptide, the fusion polypeptide includingan enzyme being capable of catalyzing the oxidation of phenolic groupsand a cellulose binding peptide; and (iii) an oxidizing agent.

According to further features in preferred embodiments of the inventiondescribed below, the lignocellulose product is selected from the groupconsisting of fiber board, particle board, flakeboard, plywood andmolded composites.

According to still further features in the described preferredembodiments the lignocellulose product is selected from the groupconsisting of paper and paperboard.

According to still further features in the described preferredembodiments the lignocellulose containing material is a cell wallpreparation derived from a genetically modified or virus infected plantor cultured plant cells expressing the fusion protein.

According to still further features in the described preferredembodiments the lignocellulose containing material is selected from thegroup consisting of vegetable fiber and wood fiber derived from agenetically modified or virus infected plant expressing the fusionpolypeptide.

According to still further features in the described preferredembodiments the lignocellulose containing material is selected from thegroup consisting of vegetable fiber and wood fiber that has previouslymade contact with an oxidising enzyme fused to a cellulose bindingpeptid.

According to still further features in the described preferredembodiments the phenolic substituent is selected from the groupconsisting of p-coumaric acid, p-coumaryl alcohol, coniferyl alcohol,sinapyl alcohol, ferulic acid p-hydroxybenzoic acid and any otherphenolic group that can be oxidized.

According to still further features in the described preferredembodiments the phenolic polymer forms an integral part of thelignocellulose containing material.

According to still further features in the described preferredembodiments the phenolic polymer is lignin.

According to still further features in the described preferredembodiments the phenolic polymer is a phenolic polysaccharide.

According to still further features in the described preferredembodiments the polysaccharide portion of the phenolic polysaccharide isselected from the group consisting of modified and unmodified starches,modified and unmodified cellulose, and modified and unmodifiedhemicelluloses.

According to still further features in the described preferredembodiments the phenolic polysaccharide is selected from the groupconsisting of ferulylated arabinoxylans and ferulylated pectins.

According to still further features in the described preferredembodiments the reaction medium is incubated for a period of from 1minute to 10 hours.

According to still further features in the described preferredembodiments the fusion polypeptide is incubated in the presence of theoxidizing agent for a period of from 1 minute to 10 hours.

According to still further features in the described preferredembodiments the enzyme is selected from the group consisting of oxidasesand peroxidases.

According to still further features in the described preferredembodiments the enzyme is an oxidase selected from the group consistingof laccases (EC 1.10.3.2), catechol oxidases (EC 1.10.3.1) and bilirubinoxidases (EC 1.3.3.5), and the oxidizing agent is oxygen.

According to still further features in the described preferredembodiments the enzyme is a laccase and is present in an amount in therange of 0.02-2000 LACU per g of dry lignocellulose.

According to still further features in the described preferredembodiments the reaction medium is aerated.

According to still further features in the described preferredembodiments the enzyme is a laccase encoded by a polynucleotide obtainedfrom a fungus of the genus Botrytis, Myceliophthora, or Trametes.

According to still further features in the described preferredembodiments the fungus is Trametes versicolor or Trametes villosa.

According to still further features in the described preferredembodiments the enzyme is a laccase from Acer pseudoplanus.

According to still further features in the described preferredembodiments the enzyme is a peroxidase and the oxidizing agent ishydrogen peroxide.

According to still further features in the described preferredembodiments the peroxidase is present in an amount in the range of0.02-2000 PODU per g of dry lignocellulose, and the initialconcentration of hydrogen peroxide in the reaction medium is in therange of 0.01-100 mM.

According to still further features in the described preferredembodiments the amount of lignocellulose employed corresponds to 0.1-90%by weight of the reaction medium, calculated as dry lignocellulose.

According to still further features in the described preferredembodiments the temperature of the reaction medium is in the range of10°-120° C.

According to still further features in the described preferredembodiments the temperature of the reaction medium is in the range of15°-90° C.

According to still further features in the described preferredembodiments an amount of the phenolic polysaccharide in the range of0.1%-10% by weight.

According to still further features in the described preferredembodiments the pH in the reaction medium is in the range of 3-10.

According to still further features in the described preferredembodiments the pH in the reaction medium is in the range of 4-9.

According to still further features in the described preferredembodiments the reaction medium further comprising a lignocellulosecontaining material devoid of the fusion protein.

According to still further features in the described preferredembodiments the lignocellulose containing material devoid of the fusionprotein is selected from the group consisting of vegetable fiber, woodfiber, wood chips, wood flakes, wood veneer and recycled fibers.

Further according to the present invention there is provided alignocellulose product obtainable by the process described herein.

According to another aspect of the present invention there is provided agenetically modified or viral infected plant or cultured plant cellsexpressing a fusion protein including an enzyme being capable ofcatalyzing the oxidation of phenolic groups and a cellulose bindingpeptide.

According to still further features in the described preferredembodiments the fusion protein being compartmentalized within cells ofthe plant or cultured plant cells, so as to be sequestered from cellwalls of the cells of the plant or cultured plant cells.

According to still further features in the described preferredembodiments expression of the fusion protein is under a control of aconstitutive or tissue specific plant promoter.

According to still further features in the described preferredembodiments the fusion protein is compartmentalized within a cellularcompartment selected from the group consisting of cytoplasm, endoplasmicreticulum, golgi apparatus, oil bodies, starch bodies, chloroplastids,chloroplasts, chromoplastids, chromoplasts, vacuole, lysosomes,mitochondria, and nucleus.

According to still another aspect of the present invention there isprovided a composition of matter comprising a cell wall preparationderived from a genetically modified or virus infected plant or culturedplant cells expressing a fusion protein including an enzyme beingcapable of catalyzing the oxidation of phenolic groups and a cellulosebinding peptide, the fusion protein being immobilized to cellulose inthe cell wall preparation via the cellulose binding peptide.

According to still another aspect of the present invention there isprovided a nucleic acid molecule comprising (a) a promoter sequence fordirecting protein expression in plant cells; and (b) a heterologousnucleic acid sequence including (i) a first sequence encoding acellulose binding peptide; and (ii) a second sequence encoding an enzymebeing capable of catalyzing the oxidation of phenolic groups, whereinthe first and second sequences are joined together in frame.

According to still further features in the described preferredembodiments the nucleic acid molecule further comprising a sequenceelement selected from the group consisting of an origin of replicationfor propagation in bacterial cells, at least one sequence element forintegration into a plant's genome, a polyadenylation recognitionsequence, a transcription termination signal, a sequence encoding atranslation start site, a sequence encoding a translation stop site,plant RNA virus derived sequences, plant DNA virus derived sequences,tumor inducing (Ti) plasmid derived sequences, a transposable elementderived sequence and a plant operative signal peptide for directing aprotein to a cellular compartment of a plant cell.

According to still further features in the described preferredembodiments the cellular compartment is selected from the groupconsisting of cytoplasm, endoplasmic reticulum, golgi apparatus, oilbodies, starch bodies, chloroplastids, chloroplasts, chromoplastids,chromoplasts, vacuole, lysosomes, mitochondria, and nucleus.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a process and compositionsfor producing a lignocellulose-based product which obviates the need forpurified enzymes which are expensive materials as is compared to otherraw materials and reagents described in the prior art for use in theprocess of manufacturing lignocellulose-based products.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is of a process and composition of matter for themanufacture of a lignocellulose-based product from a lignocellulosicmaterial, which process obviates the need for using purified enzymes.

The principles and operation of a process according to the presentinvention may be better understood with reference to the accompanyingdescriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of steps and components set forth in the followingdescription. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

The present invention thus provides a process for the manufacture of alignocellulose-based product from a lignocellulosic material. Theprocess according to the present invention is effected by mixing in areaction medium (i) a phenolic polymer substituted with a phenolichydroxy group (e.g., lignin or a polysaccharide which is substitutedwith at least substituents containing a phenolic hydroxy group); (ii) alignocellulose containing material having immobilized to a cellulosicfraction thereof a fusion polypeptide, the fusion polypeptide includingan enzyme being capable of catalyzing the oxidation of phenolic groupsand a cellulose binding peptide; and (iii) an oxidizing agent.

The order of mixing/contacting the three components is unimportant aslong as the process set-up ensures that the activated lignocellulosicmaterial and the activated phenolic polysaccharide are brought togetherin a way that enables them to react in the desired manner. Thus, forexample, the oxidizing agent may be mixed with the lignocellulosecontaining material before or after being mixed with the phenolicpolymer.

As is further detailed hereinunder, the lignocellulose containingmaterial is preferably a cell wall preparation derived from agenetically modified or virus infected plant or cultured plant cellsexpressing the fusion protein. As such, the phenolic polymer may form anintegral part of the lignocellulose containing material because cellwalls of plants contain lignin which is a phenolic polymer and thus thecell wall preparation can be made to contain lignin. In this case, andin order to prevent from the enzyme to exert its catalytic activityahead of time, the cell wall preparation may be kept under anon-oxidizing atmosphere, such as an N₂ atmosphere.

It will generally be appropriate to incubate the reaction mediumcontaining the three components for a period of at least a few minutes.An incubation time of from 1 minute to 10 hours will generally besuitable, although a period of from 1 minute to 10 hours is preferable.

As already indicated, the process of the invention is well suited to theproduction of all types of lignocellulose-based products, e.g., varioustypes of fiber board (such as hardboard), particle board, flakeboard,such as oriented-strand board (OSB), plywood, molded composites (e.g.,shaped articles based on wood particles, often in combination withother, non-lignocellulosic materials, e.g., certain plastics), paper andpaperboard (such as cardboard, linerboard and the like).

Lignocellulose Containing Material:

The lignocellulose containing material employed in the method of theinvention can be in any appropriate form, e.g., in the form of vegetablefiber (such as wood fiber) with the provision that it is derived from agenetically modified or virus infected plant expressing the fusionpolypeptide.

If appropriate, a lignocellulosic material can be used in combinationwith a non-lignocellulosic material having phenolic hydroxyfunctionalities. Using the process of the invention, intermolecularlinkages between the lignocellulosic material and thenon-lignocellulosic material, respectively, may then be formed (i.e., ina manner analogous to that in which intermolecular linkages are formedwhen lignocellulosic materials alone are employed in the process),resulting in a composite product. Besides functioning as a goodadhesive/binder, the phenolic polysaccharide also serves as a good“gap-filler”, which is a big advantage when producing, e.g., particleboards from large wood particles.

It will normally be appropriate to employ the lignocellulosic materialin question in an amount corresponding to a weight percentage of drylignocellulosic material [dry substance (DS)] in the reaction medium inthe range of 0.1-90%.

The temperature of the reaction mixture in the process of the inventionmay suitably be in the range of 10° C.-120° C., as appropriate; however,a temperature in the range of 15° C.-90° C. is generally to bepreferred. As illustrated by the working examples described in U.S. Pat.No. 5,846,788, it is anticipated that the reactions involved in aprocess of the invention may take place very satisfactorily at ambienttemperatures around 20° C.

In addition to lignocellulose containing material to which the fusionprotein is immobilized, the reaction medium according to the presentinvention may include a lignocellulose containing material devoid ofsuch fusion protein, such as, but not limited to, vegetable fiber, woodfiber, wood chips, wood flakes, wood veneer and recycled fibers.

Phenolic Polymers:

The phenolic polymers employed in the process of the invention maysuitably be materials obtainable from natural sources or polymers whichhave been chemically modified by the introduction of substituents havingphenolic hydroxy groups. Examples of the latter category are modifiedstarches containing phenolic substituents, e.g., acyl-type substituentsderived from hydroxy-substituted benzoic acids (such as, e.g., 2-, 3- or4-hydroxybenzoic acid).

The phenolic substituent(s) in phenolic polysaccharides suited for usein the context of the present invention may suitably be linked to thepolymer species by, e.g., ester linkages or ether linkages.

Very suitable phenolic polymers are phenolic polysaccharides in whichthe phenolic substituent of the phenolic polysaccharide is a substituentderived from a phenolic compound which occurs in at least one of thefollowing plant-biosynthetic pathways: from p-coumaric acid top-coumaryl alcohol, from p-coumaric acid to coniferyl alcohol and fromp-coumaric acid to sinapyl alcohol; p-coumaric acid itself and the threementioned “end products” of the latter three biosynthetic pathways arealso relevant compounds in this respect. Examples of relevant“intermediate” compounds formed in these biosynthetic pathways includecaffeic acid, ferulic acid (i.e., 4-hydroxy-3-methoxycinnamic acid),5-hydroxy-ferulic acid and sinapic acid.

Particularly suitable phenolic polysaccharides are those which exhibitgood solubility in water, and thereby in aqueous media in the context ofthe invention. In this and other respects, a number of types of phenolicpolysaccharides which are readily obtainable in uniform quality fromvegetable sources have been found to be particularly well-suited for usein the process of the present invention. These include, but are in noway limited to, phenolic arabino and heteroxylans, and phenolic pectins.Very suitable examples thereof are ferulylated arabinoxylans(obtainable, e.g., from wheat bran or maize bran) and ferulylatedpectins (obtainable from, e.g., beet pulp), i.e., arabinoxylans andpectins containing ferulyl substituents attached via ester linkages tothe polysaccharide molecules.

The amount of phenolic polysaccharide or other phenolic polymers, suchas lignin, employed in the process of the invention will generally be inthe range of 0.01-10 weight percent, based on the weight oflignocellulosic material (calculated as dry lignocellulosic material),and amounts in the range of about 0.02-6 weight percent (calculated inthis manner) will often be very suitable.

Enzymes and Polynucleotides Encoding Same

In principle, any type of enzyme capable of catalyzing oxidation ofphenolic groups may be employed in the process of the invention, withthe provision that a polynucleotide encoding same has been isolated oris readily isolateable using conventional genetic engineering isolationtechniques and which can therefore be expressed as a part of a fusionpolypeptide.

Preferred enzymes are, however, oxidases, e.g., laccases (EC 1.10.3.2),catechol oxidases (EC 1.10.3.1) and bilirubin oxidases (EC 1.3.3.5) andperoxidases (EC 1.11.1.7). In some cases it may be appropriate to employtwo or more different enzymes in the process of the invention.

Among types of oxidases (in combination with which oxygen—e.g.,atmospheric oxygen—is an excellent oxidizing agent), laccases haveproved to be well suited for use in the method of the invention.

Polynucleotides encoding laccases have been or are readily isolateablefrom a variety of plant and microbial sources, notably bacteria andfungi (including filamentous fungi and yeasts), see, for example, U.S.Pat. Nos. 5,843,745; 5,795,760; 5,770,418; and 5,750,388, which areincorporated herein by reference. Suitable examples of polynucleotidesencoding laccases include those obtained or obtainable from strains ofAspergillus, Neurospora (e.g., N. crassa), Podospora, Botrytis,Collybia, Fomes, Lentinus, Pleurotus, Trametes—some species/strains ofwhich are known by various names and/or have previously been classifiedwithin other genera; e.g. Trametes villosa=T. pinsitus=Polyporuspinsitis (also known as P. pinsitus or P. villosus)=Coriolus pinsitus,Polyporus, Rhizoctonia (e.g., R. solani), Coprinus (e.g., C.plicatilis), Psatyrella, Myceliophthora (e.g., M. thermophila),Schytalidium, Phlebia (e.g. P. radita; see WO 92/01046), or Coriolus(e.g., C. hirsutus; see JP 2-238885,).

A preferred laccase in the context of the invention is that obtainablefrom Trametes villosa or Acer pseudoplanus.

Polynucleotides encoding peroxidase enzymes (EC 1.11.1) employed in themethod of the invention are preferably those obtained or obtainable fromplants (e.g., horseradish peroxidase or soy bean peroxidase) or frommicroorganisms, such as fungi or bacteria. In this respect, somepreferred fungi include strains belonging to the sub-divisionDeuteromycotina, class Hyphomycetes, e.g., Fusarium, Humicola,Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces,Ulocladium, Embellisia, Cladosporium or Dreschlera, in particularFusarium oxysporum (DSM 2672,), Humicola insolens, Trichoderma resii,Myrothecium verrucana (IFO 6113,), Verticillum alboatrum, Verticillumdahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago,Ulocladium chartarum, Embellisia alli or Dreschlera halodes.

Other preferred fungi include strains belonging to the sub-divisionBasidiomycotina, class Basidiomycetes, e.g., Coprinus, Phanerochaete,Coriolus or Trametes, in particular Coprinus cinereus f. microsporus(IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g.,NA-12) or Trametes versicolor (e.g. PR4 28-A).

Further preferred fungi include strains belonging to the sub-divisionZygomycotina, class Mycoraceae, e.g., Rhizopus or Mucor, in particularMucor hiemalis.

Some preferred bacteria include strains of the order Actinomycetales,e.g., Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus(IFO 12382) or Streptoverticillum verticillium ssp. verticillium.

Other preferred bacteria include Bacillus pumilus (ATCC 12905), Bacillusstearothermophilus, Rhodobacter sphaeroides, Rhodomonas palustri,Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonasfluorescens (NRRL B-11).

Further preferred bacteria include strains belonging to Myxococcus,e.g., M. virescens.

Other potential sources of useful sources for polynucleotides encodingperoxidases are listed in B. C. Saunders et al., Peroxidase, London1964, pp. 41-43.

Cellulose Binding Peptides:

As used herein in the specification and in the claims section below, thephrase “cellulose binding peptide” includes peptides e.g., proteins anddomains (portions) thereof, which are capable of, when expressed inplant cells, affinity binding to a plant derived cellulosic (e.g.,lignocellulosic) matter, e.g., following homogenization and cell ruptureor during plant growth and development. The phrase thus includes, forexample, peptides which were screened for their cellulose bindingactivity out of a library, such as a peptide library or a DNA library(e.g., a cDNA expression library or a display library) and the genesencoding such peptides isolated and are expressible in plants. Yet, thephrase also includes peptides designed and engineered to be capable ofbinding to cellulose and/or units thereof.

Such peptides include amino acid sequences expressible in plants thatare originally derived from a cellulose binding region of, e.g., acellulose binding protein (CBP) or a cellulose binding domain (CBD). Thecellulose binding peptide according to the present invention can includeany amino acid sequence expressible in plants which binds to a cellulosepolymer. For example, the cellulose binding domain or protein can bederived from a cellulase, a binding domain of a cellulose bindingprotein or a protein screened for, and isolated from, a peptide library,or a protein designed and engineered to be capable of binding tocellulose or to saccharide units thereof, and which is expressible inplants. The cellulose binding domain or protein can be naturallyoccurring or synthetic, as long as it is expressible in plants. Suitablepolysaccharidases from which a cellulose binding domain or proteinexpressible in plants may be obtained include β-1,4-glucanases. In apreferred embodiment, a cellulose binding domain or protein from acellulase or scaffoldin is used. Typically, the amino acid sequence ofthe cellulose binding peptide expressed in plants according to thepresent invention is essentially lacking in the hydrolytic activity of apolysaccharidase (e.g., cellulase, chitinase), but retains the cellulosebinding activity. The amino acid sequence preferably has less than about10% of the hydrolytic activity of the native polysaccharidase; morepreferably less than about 5%, and most preferably less than about 1% ofthe hydrolytic activity of the native polysaccharidase, ideally noactivity altogether.

The cellulose binding domain or protein can be obtained from a varietyof sources, including enzymes and other proteins which bind to cellulosewhich find use in the subject invention.

In Table 4 below are listed those binding domains which bind to one ormore soluble/insoluble polysaccharides including all binding domainswith affinity for soluble glucans (α, β, and/or mixed linkages). The N1cellulose-binding domain from endoglucanase CenC of C. fimi is the onlyprotein known to bind soluble cellosaccharides and one of a small set ofproteins which are known to bind any soluble polysaccharides. Also,listed in Tables 1 to 3 are examples of proteins containing putativeβ-1,3-glucan-binding domains (Table 1); proteins containingStreptococcal glucan-binding repeats (Cpl superfamily) (Table 2); andenzymes with chitin-binding domains, which may also bind cellulose(Table 3). The genes encoding each one of the peptides listed in Tables1-4 are either isolated or can be isolated as further detailedhereinunder, and therefore, such peptides are expressible in plants.Scaffoldin proteins or portions thereof, which include a cellulosebinding domain, such as that produced by Clostridium cellulovorans(Shoseyov et al., PCT/US94/04132) can also be used as the cellulosebinding peptide expressible in plants according to the presentinvention. Several fungi, including Trichoderma species and others, alsoproduce polysaccharidases from which polysaccharide binding domains orproteins expressible in plants can be isolated. Additional examples canbe found in, for example, Microbial Hydrolysis of Polysaccharides, R. A.J. Warren, Annu. Rev. Microbiol. 1996, 50:183-212; and “Advances inMicrobial Physiology” R. K. Poole, Ed., 1995, Academic Press Limited,both are incorporated by reference as if fully set forth herein. TABLE 1Overview of proteins containing putative β-1,3 glucan-binding domainsSource (strain) Protein accession No. Ref¹ Type I B. circulans (WL-12)GLCA1 P23903/M34503/JQ0420 1 B. circulans (IAM 1165) BglHJN0772/D17519/S67033 2 Type II Actinomadura sp. (FC7) XynII U08894 3Arthrobacter sp. GLCI D23668 9 (YCWD3) O. xanthineolytica GLCP22222/M60826/A39094 4 R. faecitabidus RP I Q05308/A45053/D10753 5a, b(YLM-50) R. communis Ricin A12892 6 S. lividans (1326) XlnAP26514/M64551/JS07986 7 T. tridentatus FactorGa D16622 8B.: Bacillus,O.: Oerskovia,R. faecitabidus: Rarobacter faecitabidus,R. communis: Ricinus communis,S.: Streptomyces,T.: Tachypleus (Horseshoe Crab)¹References:1) Yahata et al. (1990) Gene 86, 113-1172) Yamamoto et al. (1993) Biosci. Biotechnol. Biochem. 57, 1518-15253) Harpin et al. (1994) EMBL Data Library4) Shen et al. (1991) J. Biol. Chem. 266, 1058-10635a) Shimoi et al. (1992) J. Biol. Chem. 267, 25189-251955b) Shimoi et al. (1992) J. Biochem 110, 608-6136) Horn et al. (1989) Patent A128927) Shareck et al. (1991) Gene 107, 75-828) Seki et al. (1994) J. Biol. Chem. 269, 1370-13749) Watanabe et al. (1993) EMBL Data Library

TABLE 2 Overview of proteins containing Streptococcal glucan-bindingrepeats (Cpl superfamily) Source Protein Accession No. Ref.² S. downei(sobrinus) (0MZ176) GTF-I D13858  1 S. downei (sobrinus) (MFe28) GTF-IP11001/M17391  2 S. downei (sobrinus) (MFe28) GTF-S P29336/M30943/A41483 3 S. downei (sobrinus) (6715) GTF-I P27470/D90216/A38175  4 S. downei(sobrinus) DEI L34406  5 S. mutants (Ingbritt) GBP M30945/A37184  6 S.mutants (GS-5) GTF-B A33128  7 S. mutants (GS-5) GTF-BP08987/M17361/B33135  8 S. mutants GTF-B^(3′-ORF) P05427/C33135  8 S.mutants (GS-5) GTF-C P13470/M17361/M22054  9 S. mutants (GS-5) GTF-C notavailable 10 S. mutants (GS-5) GTF-D M29296/A45866 11 S. salivariusGTF-J A44811/S22726/S28809 12 Z11873/M64111 S. salivarius GTF-KS22737/S22727/Z11872 13 S. salivarius (ATCC25975) GTF-L L35495 14 S.salivarius (ATCC25975) GTF-M L35928 14 S. pneumoniae R6 LytAP06653/A25634/M13812 15 S. pneumoniae PspA A41971/M74122 16 Phage HB-3HBL P32762/M34652 17 Phage Cp-1 CPL-1 P15057/J03586/A31086 18 Phage Cp-9CPL-9 P19386/M34780/JQ0438 19 Phage EJ-1 EJL A42936 20 C. difficile (VPI10463) ToxA P16154/A37052/M30307 21 X51797/S08638 C. difficile (BARTSW1) ToxA A60991/X17194 22 C. difficile (VPI 10463) ToxBP18177/X53138/X6098 23, 24 S10317 C. difficile (1470) ToxB S44271/Z2327725, 26 C. novyi α-toxin S44272/Z23280 27 C. novyi α-toxin Z48636 28 C.acetobutylicum (NCIB8052) CspA S49255/Z37723 29 C. acetobutylicum(NCIB8052) CspB Z50008 30 C. acetobutylicum (NCIB8052) CspC Z50033 30 C.acetobutylicum (NCIB8052) CspD Z50009 30²References:1) Sato et al. (1993) DNA sequence 4, 19-272) Ferreti et al. (1987) J. Bacteriol. 169, 4271-42783) Gilmore et al. (1990) J. Infect. Immun. 58, 2452-24584) Abo et al. (1991) J. Bacteriol. 173, 989-9965) Sun et al. (1994) J. Bacteriol. 176, 7213-72226) Banas et al. (1990) J. Infect. Immun. 58, 667-6737) Shiroza et al. (1990) Protein Sequence Database8) Shiroza et al. (1987) J. Bacteriol. 169, 4263-42709) Ueda et al. (1988) Gene 69, 101-10910) Russel (1990) Arch. Oral. Biol. 35, 53-5811) Honda et al. (1990) J. Gen. Microbiol. 136, 2099-210512) Giffard et al. (1991) J. Gen. Microbiol. 137, 2577-259313) Jacques (1992) EMBL Data Library14) Simpson et al. (1995) J. Infect. Immun. 63, 609-62115) Gargia et al. (1986) Gene 43, 265-27216) Yother et al. (1992) J. Bacteriol. 174, 601-60917) Romero et al. (1990) J. Bacteriol. 172, 5064-507018) Garcia et al. (1988) Proc. Natl. Acad. Sci, USA 85, 914-91819) Garcia et al. (1990) Gene 86, 81-8820) Diaz et al. (1992) J. Bacteriol. 174, 5516-552521) Dove et al. (1990) J. Infect. Immun. 58, 480-48822) Wren et al. (1990) FEMS Microbiol. Lett. 70, 1-623) Barroso et a. (1990) Nucleic Acids Res. 18, 4004-400424) von Eichel-Streiber et al. (1992) Mol. Gen. Genet. 233, 260-26825) Sartinger et al. (1993) EMBL Data Library26) von Eichel-Streiber et al. (1995) Mol. Microbiol. In Press27) Hofmann et al. (1993) EMBL Data Library28) Hofmann et al. (1995) Mol. Gen. Genet. In Press29) Sanchez et al. (1994) EMBL Data Library30) Sanchez et al. (1995) EMBL Data Library

New cellulose binding peptides with interesting binding characteristicsand specificities can be identified and screened for and the genesencoding same isolated using well known molecular biology approachescombined with a variety of other procedures including, for example,spectroscopic (titration) methods such as: NMR spectroscopy (Zhu et al.Biochemistry (1995) 34:13196-13202, Gehring et al. Biochemistry (1991)30:5524-5531), UV difference spectroscopy (Belshaw et al. Eur. J.Biochem. (1993) 211:717-724), fluorescence (titration) spectroscopy(Miller et al. J. Biol. Chem. (1983) 258:13665-13672), UV orfluorescence stopped flow analysis (De Boeck et al. Eur. J. Biochem.(1985) 149:141-415), affinity methods such as affinity electrophoresis(Mimura et al. J. chromatography (1992) 597:345-350) or affinitychromatography on immobilized mono or oligosaccharides, precipitation oragglutination analysis including turbidimetric or nephelometric analysis(Knibbs et al. J. Biol. Chem. (1993) 14940-14947), competitiveinhibition assays (with or without quantitative IC50 determination) andvarious physical or physico-chemical methods including differentialscanning or isothermal titration calorimetry (Sigurskjold et al. J.Biol. Chem. (1992) 267:8371-8376; Sigurskjold et al. Eur. J. Biol.(1994) 225:133-141) or comparative protein stability assays (melts) inthe absence or presence of oligo saccharides using thermal CD orfluorescence spectroscopy.

The K_(a) for binding of the cellulose binding domains or proteins tocellulose is at least in the range of weak antibody-antigen extractions,i.e., 10³, preferably 10⁴, most preferably 10⁶ M⁻¹. If the binding ofthe cellulose binding domain or protein to cellulose is exothermic orendothermic, then binding will increase or decrease, respectively, atlower temperatures, providing a means for temperature modulation of thebinding step. TABLE 3 Overview of enzymes with chitin-binding domainsSource (strain) Enzyme Accession No. Ref.³ Bacterial enzymes Type IAeromonas sp. (No10S-24) Chi D31818  1 Bacillus circulans (WL-12) ChiA1P20533/M57601/A38368  2 Bacillus circulans (WL-12) ChiD P27050/D10594  3Janthinobacterium lividum Chi69 U07025  4 Streptomyces griseus ProteaseC A53669  5 Type II Aeromonas cavia (K1) Chi U09139  6 Alteromonas sp(0-7) Chi85 A40633/P32823/D13762  7 Autographa californica (C6)NPH-128^(a) P41684/L22858  8 Serratia marcescens ChiAA25090/X03657/L01455/P07254  9 Type III Rhizopus oligosporus (IFO8631)Chi1 P29026/A47022/D10157/S27418 10 Rhizopus oligosporus (IFO8631) Chi2P29027/B47022/D10158/S27419 10 Saccharomyces cerevisiae ChiS50371/U17243 11 Saccharomyces cerevisiae Chi1 P29028/M74069 12 (DBY939)Saccharomyces cerevisiae Chi2 P29029/M7407/B41035 12 (DBY918) Plantenzymes Hevein superfamily Allium sativum Chi M94105 13 Amaranthuscaudatus AMP-1^(b) P27275/A40240 14, 15 Amaranthus caudatus AMP-2^(b)S37381/A40240 14, 15 Arabidopsis thaliana ChiB P19171/M38240/B45511 16(cv. colombia) Arabidopsis thaliana PHP^(c) U01880 17 Brassica napus ChiU21848 18 Brassica napus Chi2 Q09023/M95835 19 Hevea brasiliensisHev1^(d) P02877/M36986/A03770/A38288 20, 21 Hordeum vulgare Chi33 L3421122 Lycopersicon esculentum Chi9 Q05538/Z15140/S37344 23 Nicotianatabacum CBP20^(e) S72424 24 Nicotiana tabacum Chi A21091 25 Nicotianatabacum (cv. Havana) Chi A29074/M15173/S20981/S19855 26 Nicotianatabacum (FB7-1) Chi JQ0993/S0828 27 Nicotiana tabacum (cv. Samsun) ChiA16119 28 Nicotiana tabacum (cv. Havana) Chi P08252/X16939/S08627 27Nicotiana tabacum (cv. BY4) Chi P24091/X51599/X64519//S13322 26, 27, 29Nicotiana tabacum (cv. Havana) Chi P29059/X64518/S20982 26 Oryza sativum(IR36) ChiA L37289 30 Oryza sativum ChiB JC2253/S42829/Z29962 31 Oryzasativum Chi S39979/S40414/X56787 32 Oryza sativum (cv. Japonicum) ChiX56063 33 Oryza sativum (cv. Japonicum) Chi1 P24626/X54367/S14948 34Oryza sativum Chi2 P25765/S15997 35 Oryza sativum (cv. Japonicum) Chi3D16223 Oryza sativum ChiA JC2252/S42828 30 Oryza sativum Chi1 D16221 32Oryza sativum (IR58) Chi U02286 36 Oryza sativum Chi X87109 37 Pisumsativum (cv. Birte) Chi P36907/X63899 38 Pisum sativum (cv. Alcan) Chi2L37876 39 Populus trichocarpa Chi S18750/S18751/X59995/P29032 40 Populustrichocarpa (H11-11) Chi U01660 41 Phaseolus vulgaris (cv. Saxa) ChiA24215/S43926/Jq0965/P36361 42 Phaseolus vulgaris (cv. Saxa) ChiP06215/M13968/M19052/A25898 43, 44, 45 Sambucus nigra PR-3^(f) Z46948 46Secale cereale Chi JC2071 47 Solanum tuberosum ChiB1 U02605 48 Solanumtuberosum ChiB2 U02606 48 Solanum tuberosum ChiB3 U02607/S43317 48Solanum tuberosum ChiB4 U02608 48 Solanum tuberosum WIN-1^(g)P09761/X13497/S04926 49 (cv. Maris Piper) Solanum tuberosum WIN-2^(g)P09762/X13497/S04927 49 (cv. Maris Piper) Triticum aestivum ChiS38670/X76041 50 Triticum aestivum WGA-1^(h) P10968/M25536/S09623/S0728951, 52 Triticum aestivum WGA-2^(h) P02876/M25537/S09624 51, 53 Triticumaestivum WGA-3^(h) P10969/J02961/S10045/A28401 54 Ulmus americana(NPS3-487) Chi L22032 55 Urtica dioica AGL^(i) M87302 56 Vignaunguiculata Chi1 X88800 57 (cv. Red caloona)^(a)NHP: nuclear polyhedrosis virus endochitinase like sequence; Chi:chitinase,^(b)anti-microbial peptide,^(c)pre-hevein like protein,^(d)hevein,^(e)chitin-binding protein,^(f)pathogenesis related protein,^(g)wound-induced protein,^(h)wheat germ agglutinin,^(i)agglutinin (lectin).³References:1) Udea et al. (1994) J. Ferment. Bioeng 78, 205-2112) Watanabe et al. (1990) J. Biol. Chem. 265, 15659-165653) Watanabe et al. (1992) J. Bacteriol. 174, 408-4144) Gleave et al. (1994) EMBL Data Library5) Sidhu et al. (1994) J. Biol. Chem. 269, 20167-201716) Jones et al. (1986) EMBO J. 5, 467-4737) Sitrit et al. (1994) EMBL Data Library8) Genbank entry only9) Tsujibo et al. (1993) J. Bacteriol. 175, 176-18110) Yanai et al. (1992) J. Bacteriol. 174, 7398-740611) Pauley (1994) EMBL Data Library12) Kuranda et al. (1991) J. Biol. Chem. 266, 19758-1976713) van Damme et al. (1992) EMBL Data Library14) Broekaert et al. (1992) Biochemistry 31, 4308-431415) de Bolle et al. (1993) Plant Mol. Physiol. 22, 1187-119016) Samac et al. (1990) Plant Physiol. 93, 907-91417) Potter et al. (1993) Mol. Plant Microbe Interact. 6, 680-68518) Buchanan-Wollaston (1995) EMBL Data Library19) Hamel et al. (1993) Plant Physiol. 101, 1403-140320) Broekaert et al. (1990) Proc. Natl. Acad. Sci. USA 87, 7633-763721) Lee et al. (1991) J. Biol. Chem. 266, 15944-1594822) Leah et al. (1994) Plant Physiol. 6, 579-58923) Danhash et al. (1993) Plant Mol. Biol. 22 1017-102924) Ponstein et al. (1994) Plant Physiol. 104, 109-11825) Meins et al. (1991) Patent EP0418695-A126) van Buuren et al. (1992) Mol. Gen. Genet. 232, 460-46927) Shinshi et al. (1990) Plant Mol. Biol. 14, 357-36828) Cornellisen et al. (1991) Patent EP0440304-A229) Fukuda et al. (1991) Plant Mol. Biol. 16, 1-1030) Yun et al. (1994) EMBL Data Library31) Kim et al. (1994) Biosci. Biotechnol. Biochem. 58, 1164-116632) Nishizawa et al. (1993) Mol. Gen. Genet. 241, 1-1033) Nishizawa et al. (1991) Plant Sci 76, 211-21834) Huang et al. (1991) Plant Mol. Biol. 16, 479-48035) Zhu et al. (1991) Mol. Gen. Genet. 226, 289-29636) Muthukrishhnan et al. (1993) EMBL Data Library37) Xu (1995) EMBL Data Library38) Vad et al. (1993) Plant Sci 92, 69-7939) Chang et al. (1994) EMBL Data Library40) Davis et al. (1991) Plant Mol. Biol. 17, 631-63941) Clarke et al. (1994) Plant Mol. Biol 25, 799-81542) Broglie et al. (1989) Plant Cell 1, 599-60743) Broglie et al. (1986) Proc. Natl. acad Sci. USA 83, 6820-682444) Lucas et al. (1985) FEBS Lett. 193, 208-21045) Hedrick et al. (1988) Plant Physiol. 86, 182-18646) Roberts et al. (1994) EMBL Data LibraryI47) Vamagami et al. (1994) Biosci. Biotechnol. Biochem. 58, 322-32948) Beerhues et al. (1994) Plant Mol. Biol. 24, 353-36749) Stanford et al. (1989) Mol. Gen. Genet. 215, 200-20850) Liao et al. (1993) EMBL Data Library51) Smith et al. (1989) Plant Mol. Biol. 13, 601-60352) Wright et al. (1989) J. Mol. Evol. 28, 327-33653) Wright et al. (1984) Biochemistry 23, 280-28754) Raikhel et al. (1987) Proc. Natl. acad Sci. USA 84, 6745-674955) Hajela et al. (1993) EMBL Data Library56) Lerner et al. (1992) J. Biol. Chem. 267, 11085-1109157) Vo et al. (1995) EMBL Data Library

TABLE 4 Sources of polysaccharide binding domains Proteins Where BindingBinding Domain Domain is Found Cellulose Binding β-glucanases(avicelases, CMCases, Domains¹ cellodextrinases) exoglucanses orcellobiohydrolases cellulose binding proteins xylanases mixedxylanases/glucanases esterases chitinases β-1,3-glucanasesβ-1,3-(β-1,4)-glucanases (β-)mannanases β-glucosidases/galactosidasescellulose synthases (unconfirmed) Starch/Maltodextrin -amylases^(2,3)Binding Domains β-amylases^(4,5) pullulanases glucoamylases^(6,7)cyclodextrin glucotransferases⁸⁻¹⁰ (cyclomaltodextringlucanotransferases) maltodextrin binding proteins¹¹ Dextran BindingDomains (Streptococcal) glycosyl transferases¹² dextran sucrases(unconfirmed) Clostridial toxins^(13,14) glucoamylases⁶ dextran bindingproteins β-Glucan Binding Domains β-1,3-glucanases^(15,16)β-1,3-(β-1,4)-glucanases (unconfirmed) β-1,3-glucan binding protein¹⁷Chitin Binding Domains chitinases chitobiases chitin binding proteins(see also cellulose binding domains) Heivein¹Gilkes et al., Adv. Microbiol Reviews, (1991) 303-315.²S?gaard et al., J. Biol. Chem. (1993) 268: 22480.³Weselake et al., Cereal Chem. (1983) 60: 98.⁴Svensson et al., J. (1989) 264: 309.⁵Jespersen et al., J. (1991) 280: 51.⁶Belshaw et al., Eur. J. Biochem. (1993) 211: 717.⁷Sigurskjold et al., Eur. J. Biochem. (1994) 225: 133.⁸Villette et al, Biotechnol. Appl. Biochem. (1992) 16: 57.⁹Fukada et al., Biosci. Biotechnol. Biochem. (1992) 56: 556.¹⁰Lawson et al., J. Mol. Biol. (1994) 236: 590.¹⁴von Eichel-Streiber et al., Mol. Gen. Genet. (1992) 233: 260.¹⁵Klebl et al., J. Bacteriol. (1989) 171: 6259.¹⁶Watanabe et al., J. Bacteriol. (1992) 174: 186.¹⁷Duvic et al., J. Biol. Chem. (1990): 9327.

Thus, and as already stated, the phrase “polysaccharide binding peptide”includes an amino acid sequence which comprises at least a functionalportion of a polysaccharide binding region (domain) of apolysaccharidase or a polysaccharide binding protein. The phrase furtherrelates to a polypeptide screened for its cellulose binding activity outof a library, such as a peptide library or a DNA library (e.g., a cDNAlibrary or a display library). By “functional portion” is intended anamino acid sequence which binds to cellulose.

The techniques used in isolating polysaccharidase genes, such ascellulase genes, and genes for cellulose binding proteins are known inthe art, including synthesis, isolation from genomic DNA, preparationfrom cDNA, or combinations thereof. (See, U.S. Pat. Nos. 5,137,819;5,202,247; 5,340,731; 5,496,934; and 5,837,814). The sequences forseveral binding domains, which bind to soluble oligosaccharides areknown (See, FIG. 1 of PCT/CA97/00033, WO 97/26358). The DNAs coding fora variety of polysaccharidases and polysaccharide binding proteins arealso known. Various techniques for manipulation of genes are well known,and include restriction, digestion, resection, ligation, in vitromutagenesis, primer repair, employing linkers and adapters, and the like(see Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989, which is incorporatedherein by reference).

The amino acid sequence of a polysaccharidase can be used to design aprobe to screen a cDNA or a genomic library prepared from mRNA or DNAfrom cells of interest as donor cells for a polysaccharidase gene or apolysaccharide binding protein gene. By using the polysaccharidase cDNAor binding protein cDNA or a fragment thereof as a hybridization probe,structurally related genes found in other species can be easily clonedand provide a cellulose binding peptide which is expressible in plantsaccording to the present invention. Particularly contemplated is theisolation of genes from organisms that express polysaccharidase activityusing oligonucleotide probes based on the nucleotide sequences of genesobtainable from an organism wherein the catalytic and binding domains ofthe polysaccharidase are discrete, although other polysaccharide bindingproteins also can be used (see, for example, Shoseyov, et al., Proc.Nat'l. Acad. Sci. (USA) (1992) 89:3483-3487).

Probes developed using consensus sequences for the binding domain of apolysaccharidase or polysaccharide-binding protein are of particularinterest. The β-1,4-glycanases from C. fimi characterized to date areendoglucanases A, B, C and D (CenA, CenB, CenC and CenD, respectively),exocellobiohydrolases A and B (CbhA and CbhB, respectively), andxylanases A and D (Cex and XylD, respectively) (see Wong et al. (1986)Gene, 44:315; Meinke et al. (1991) J. Bacteriol., 173:308; Coutinho etal., (1991) Mol. Microbiol. 5:1221; Meinke et al., (1993) Bacteriol.,175:1910; Meinke et al., (1994) Mol. Microbiol., 12:413; Shen et al.,Biochem. J., in press; O'Neill et al., (1986) Gene, 44:325; andMillward-Sadler et al., (1994) Mol. Microbiol., 11:375). All are modularproteins of varying degrees of complexity, but with two features incommon: a catalytic domain (CD) and a cellulose-binding domain (CBD)which can function independently (see Millward-Sadler et al., (1994)Mol. Microbiol., 11:375; Gilkes et al., (1988) J. Biol. Chem.,263:10401; Meinke et al., (1991) J. Bacteriol., 173:7126; and Coutinhoet al., (1992) Mol. Microbiol., 6:1242). In four of the enzymes, CenB,CenD, CbhA and CbhB, fibronectin type III (Fn3) repeats separate theN-terminal CD from the C-terminal CBD. The CDs of the enzymes come fromsix of the families of glycoside hydrolases (see Henrissat (1991)Biochem. J., 280:309; and Henrissat et al., (1993) Biochem. J.,293:781); all of the enzymes have an N- or C-terminal CBD or CBDs (seeTomme et al., Adv. Microb. Physiol., in press); CenC has tandem CBDsfrom family IV at its N-terminus; CenB and XylD each have a second,internal CBD from families III and II, respectively. Cex and XylD areclearly xylanases; however, Cex, but not XylD, has low activity oncellulose. Nonetheless, like several other bacterial xylanases (seeGilbert et al., (1993) J. Gen. Microbiol., 139:187), they have CBDs. C.fimi probably produces other β-1,4-glycanases. Similar systems areproduced by related bacteria (see Wilson (1992) Crit. Rev. Biotechnol.,12:45; and Hazlewood et al., (1992) J. Appl. Bacteriol., 72:244).Unrelated bacteria also produce glycanases; Clostridium thermocellum,for example, produces twenty or more β-1,4-glycanases (see Beguin etal., (1992) FEMS Microbiol. Lett., 100:523). The CBD derived from C.fimi endoglucanase C N1, is the only protein known to bind solublecellosaccharides and one of a small set of proteins that are known tobind any soluble polysaccharides.

Examples of suitable binding domains are shown in FIG. 1 ofPCT/CA97/00033 (WO 97/26358), which presents an alignment of bindingdomains from various enzymes that bind to polysaccharides and identifiesamino acid residues that are conserved among most or all of the enzymes.This information can be used to derive a suitable oligonucleotide probeusing methods known to those of skill in the art. The probes can beconsiderably shorter than the entire sequence but should at least be 10,preferably at least 14, nucleotides in length. Longer oligonucleotidesare useful, up to the full length of the gene, preferably no more than500, more preferably no more than 250, nucleotides in length. RNA or DNAprobes can be used. In use, the probes are typically labeled in adetectable manner, for example, with ³²P, ³H, biotin, avidin or otherdetectable reagents, and are incubated with single-stranded DNA or RNAfrom the organism in which a gene is being sought. Hybridization isdetected by means of the label after the unhybridized probe has beenseparated from the hybridized probe. The hybridized probe is typicallyimmobilized on a solid matrix such as nitrocellulose paper.Hybridization techniques suitable for use with oligonucleotides are wellknown to those skilled in the art. Although probes are normally usedwith a detectable label that allows easy identification, unlabeledoligonucleotides are also useful, both as precursors of labeled probesand for use in methods that provide for direct detection ofdouble-stranded DNA (or DNA/RNA). Accordingly, the term “oligonucleotideprobe” refers to both labeled and unlabeled forms.

Generally, the binding domains identified by probing nucleic acids froman organism of interest will show at least about 40% identity (includingas appropriate allowances for conservative substitutions, gaps forbetter alignment and the like) to the binding region or regions fromwhich the probe was derived and will bind to a soluble β-1,4 glucan witha K_(a) of ≧10³ M⁻¹. More preferably, the binding domains will be atleast about 60% identical, and most preferably at least about 70%identical to the binding region used to derive the probe. The percentageof identity will be greater among those amino acids that are conservedamong polysaccharidase binding domains. Analyses of amino acid sequencecomparisons can be performed using programs in PC/Gene (IntelliGenetics,Inc.). PCLUSTAL can be used for multiple sequence alignment andgeneration of phylogenetic trees.

In order to isolate the polysaccharide binding protein or apolysaccharide binding domain from an enzyme or a cluster of enzymesthat binds to a polysaccharide, several genetic approaches can be used.One method uses restriction enzymes to remove a portion of the gene thatcodes for portions of the protein other than the binding portionthereof. The remaining gene fragments are fused with expression controlsequences to obtain a mutated gene that encodes a truncated protein.Another method involves the use of exonucleases such as Bal31 tosystematically delete nucleotides either externally from the 5′ and the3′ ends of the DNA or internally from a restricted gap within the gene.These gene deletion methods result in a mutated gene encoding ashortened protein molecule which can then be evaluated for substrate orpolysaccharide binding ability.

Any cellulose binding protein or cellulose binding domain may be used inthe present invention. The term “cellulose binding protein” (“CBP”)refers to any protein or polypeptide which specifically binds tocellulose. The cellulose binding protein may or may not have celluloseor cellulolytic activity. The term “cellulose binding domain” (“CBD”)refers to any protein or polypeptide which is a region or portion of alarger protein, said region or portion binds specifically to cellulose.The cellulose binding domain (CBD) may be a part or portion of acellulase, xylanase or other polysaccharidase, e.g., a chitinase, etc.,a sugar binding protein such as maltose binding protein, or scaffoldinsuch as CbpA of Clostridium celluvorans, etc. Many cellulases andhemicellulases (e.g., xylanases and mannases) have the ability toassociate with cellulose. These enzymes typically have a catalyticdomain containing the active site for substrate hydrolysis and acarbohydrate-binding domain or cellulose-binding domain for bindingcellulose. The CBD may also be from a non-catalytic polysaccharidebinding protein. To date, more than one hundred cellulose-bindingdomains (CBDs) have been classified into at least thirteen familiesdesignated I-XIII (Tomme et al. (1995) “CelluloseBinding Domains:Classification and Properties”, in ACS Symposium Series 618 EnzymaticDegradation and Insoluble Carbohydrates, pp. 142-161, Saddler and Pennereds., American Chemical Society, Washington, D.C. (Tomme I); Tomme etal. Adv. Microb. Physiol. (1995) 37:1 (Tomme II); and Smant et al.,Proc. Natl. Acad. Sci U.S.A. (1998) 95:4906,-4911, all of which areincorporated herein by reference). Any of the CBDs described in Tomme Ior II or any variants thereof, any other presently known CBDs or any newCBDs which may be identified can be used in the present invention. As anillustrative, but in no way limiting example, the CBP or CBD can be froma bacterial, ftingal, slime mold, or nematode protein or polypeptide.For a more particular illustrative example, the CBD is obtainable fromClostridium cellulovorans, Clostridium cellulovorans, or Cellulomonasfimi (e.g., CenA, CenB, CenD, Cex). In addition, the CBD may be selectedfrom a phage display peptide or peptidomimetic library, random orotherwise, using e.g., cellulose as a screening agent. (See SmithScience (1985) 228:1315-1317 and Lam, Nature (1991) 354:82-84).

Furthermore, the CBD may be derived by mutation of a portion of aprotein or polypeptide which binds to a polysaccharide other thancellulose (or hemicellulose) but also binds cellulose, such as achitinase, which specifically binds chitin, or a sugar binding proteinsuch as maltose binding protein, rendering said portion capable ofbinding to cellulose. In any event, the CBD binds cellulose orhemicellulose. Shoseyov and Doi (Proc. Natl. Acad. Sci. USA (1990)87:2192-2195) isolated a unique cellulose-binding protein (CbpA) fromthe cellulose “complex” of the cellulolytic bacterium Clostridiumcellulovorans. This major subunit of the cellulose complex was found tobind to cellulose, but had no hydrolytic activity, and was essential forthe degradation of crystalline cellulose. The CbpA gene has been clonedand sequenced (Shoseyov et al. Proc. Natl. Acad. Sci. USA (1992)89:3483-3487). Using PCR primers flanking the cellulose-binding domainof CbpA, the latter was successfully cloned into an overexpressionvector that enabled overproduction of the approximately 17 kDa CBD inEscherichia coli. The recombinant CBD exhibits very strong affinity tocellulose and chitin (U.S. Pat. No. 5,496,934; Goldstein et al., J.Bacteriol. (1993) 175:5762; PCT International Publication WO 94/24158,all are incorporated by reference as if fully set forth herein).

In recent years, several CBDs have been isolated from different sources.Most of these have been isolated from proteins that have separatecatalytic, i.e., cellulose and cellulose binding domains, and only twohave been isolated from proteins that have no apparent hydrolyticactivity but possess cellulose-binding activity (Goldstein et al. J.Bacteriol. (1993) 175:5762-5768; Morag et al. Appl. (1995) Environ.Microbiol. 61:1980-1986).

Cellulose Binding Peptide-Recombinant Protein Fusions:

The fusion of two proteins for which genes has been isolated, such as acellulose binding peptide and an oxidase, such as a laccase, is wellknown and regularly practiced in the art. Such fusion involves thejoining together of heterologous nucleic acid sequences, in frame, suchthat translation thereof results in the generation of a fused proteinproduct or a fusion proteins. Methods, such as the polymerase chainreaction (PCR), restriction, nuclease digestion, ligation, syntheticoligonucleotides synthesis and the like are typically employed invarious combinations in the process of generating fusion geneconstructs. One ordinarily skilled in the art can readily form suchconstructs for any pair or more of individual proteins. Interestingly,in most cases where such fusion or chimera proteins are produced, and inall cases where one of the proteins was a cellulose binding peptide,both the former and the latter retained their catalytic activity orfunction. In any case, an in frame spacer can be included. The lengththereof may range, for example, from several to several dozens of aminoacids. Such a spacer may also function to reduce mobilizationconstraints.

For example, Greenwood et al. (1989, FEBS Lett. 224:127-131) fused thecellulose binding region of Cellulomonas fimi endoglucanase to theenzyme alkaline phosphatase. The recombinant fusion protein retainedboth its phosphatase activity and the ability to bind to cellulose. Formore descriptions of cellulose binding fusion proteins, see U.S. Pat.No. 5,137,819 issued to Kilbum et al., and U.S. Pat. No. 5,719,044issued to Shoseyov et al. both incorporated by reference herein. Seealso U.S. Pat. No. 5,474,925. All of which are incorporated herein byreference.

Thus, according to the present invention there is provided a nucleicacid molecule comprising a promoter sequence for directing proteinexpression in plant cells and a heterologous nucleic acid sequenceincluding a first sequence encoding a cellulose binding peptide; and asecond sequence encoding an enzyme being capable of catalyzing theoxidation of phenolic groups, wherein the first and second sequences arejoined together in frame.

According to a preferred embodiment of the invention the nucleic acidmolecule further comprising a sequence element selected from the groupconsisting of an origin of replication for propagation in bacterialcells, at least one sequence element for integration into a plant'sgenome, a polyadenylation recognition sequence, a transcriptiontermination signal, a sequence encoding a translation start site, asequence encoding a translation stop site, plant RNA virus derivedsequences, plant DNA virus derived sequences, tumor inducing (Ti)plasmid derived sequences, a transposable element derived sequence and aplant operative signal peptide for directing a protein to a cellularcompartment of a plant cell.

According to still a preferred embodiment, the cellular compartment isselected from the group consisting of cytoplasm, endoplasmic reticulum,golgi apparatus, oil bodies, starch bodies, chloroplastids,chloroplasts, chromoplastids, chromoplasts, vacuole, lysosomes,mitochondria, and nucleus.

Genetically Modified Plant Material:

The present invention employs recombinant nucleic acid molecules. Such amolecule includes, for example, a promoter sequence for directingprotein expression in plant cells; and a heterologous nucleic acidsequence as further detailed herein, wherein, the heterologous nucleicacid sequence is down stream the promoter sequence, such that expressionof the heterologous nucleic acid sequence is effectable by the promotersequence. Such a nucleic acid molecule needs to be effectivelyintroduced into plant cells, so as to genetically modify the plant.

There are various methods of introducing foreign genes into bothmonocotyledonous and dicotyledenous plants (Potrykus, I., Annu. Rev.Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al.,Nature (1989) 338:274-276). The principle methods of causing stableintegration of exogenous DNA into plant genomic DNA include two mainapproaches:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev.Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes, eds. Schell, J., and Vasil, L. K., Academic Publishers, SanDiego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

(ii) direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; or by the direct incubation of DNA with germinatingpollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds.Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London,(1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986)83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. Horsch et al. in Plant Molecular Biology Manual A5,Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. The Agrobacteriumsystem is especially viable in the creation of transgenic dicotyledenousplants.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, the protoplasts are briefly exposed to a strongelectric field. In microinjection, the DNA is mechanically injecteddirectly into the cells using very small micropipettes. In microparticlebombardment, the DNA is adsorbed on microprojectiles such as magnesiumsulfate crystals or tungsten particles, and the microprojectiles arephysically accelerated into cells or plant tissues.

Following transformation plant propagation is exercised. The most commonmethod of plant propagation is by seed. Regeneration by seedpropagation, however, has the deficiency that due to heterozygositythere is a lack of uniformity in the crop, since seeds are produced byplants according to the genetic variances governed by Mendelian rules.Basically, each seed is genetically different and each will grow withits own specific traits. Therefore, it is preferred that the transgenicplant be produced such that the regenerated plant has the identicaltraits and characteristics of the parent transgenic plant, e.g., areproduction of the fusion protein. Therefore, it is preferred that thetransgenic plant be regenerated by micropropagation which provides arapid, consistent reproduction of the transgenic plants.

Micropropagation is a process of growing new generation plants from asingle piece of tissue that has been excised from a selected parentplant or cultivar. This process permits the mass reproduction of plantshaving the preferred tissue expressing the fusion protein. The newgeneration plants which are produced are genetically identical to, andhave all of the characteristics of, the original plant.

Micropropagation allows mass production of quality plant material in ashort period of time and offers a rapid multiplication of selectedcultivars in the preservation of the characteristics of the originaltransgenic or transformed plant. The advantages of cloning plants arethe speed of plant multiplication and the quality and uniformity ofplants produced. Micropropagation is a multi-stage procedure thatrequires alteration of culture medium or growth conditions betweenstages. Thus, the micropropagation process involves four basic stages:Stage one, initial tissue culturing; stage two, tissue culturemultiplication; stage three, differentiation and plant formation; andstage four, greenhouse culturing and hardening. During stage one,initial tissue culturing, the tissue culture is established andcertified contaminant-free. During stage two, the initial tissue cultureis multiplied until a sufficient number of tissue samples are producedto meet production goals. During stage three, the tissue samples grownin stage two are divided and grown into individual plantlets. At stagefour, the transgenic plantlets are transferred to a greenhouse forhardening where the plants' tolerance to light is gradually increased sothat it can be grown in the natural environment.

The basic bacterial/plant vector construct will preferably provide abroad host range prokaryote replication origin; a prokaryote selectablemarker; and, for Agrobacterium transformations, T DNA sequences forAgrobacterium-mediated transfer to plant chromosomes. Where theheterologous sequence is not readily amenable to detection, theconstruct will preferably also have a selectable marker gene suitablefor determining if a plant cell has been transformed. A general reviewof suitable markers for the members of the grass family is found inWilmink and Dons, Plant Mol. Biol. Reptr. (1993) 11:165-185.

Sequences suitable for permitting integration of the heterologoussequence into the plant genome are also recommended. These might includetransposon sequences and the like for homologous recombination as wellas Ti sequences which permit random insertion of a heterologousexpression cassette into a plant genome.

Suitable prokaryote selectable markers include resistance towardantibiotics such as ampicillin or tetracycline. Other DNA sequencesencoding additional functions may also be present in the vector, as isknown in the art.

The constructs of the subject invention will include an expressioncassette for expression of the fusion protein of interest. Usually,there will be only one expression cassette, although two or more arefeasible. The recombinant expression cassette will contain in additionto the heterologous sequence one or more of the following sequenceelements, a promoter region, plant 5′ untranslated sequences, initiationcodon depending upon whether or not the structural gene comes equippedwith one, and a transcription and translation termination sequence.Unique restriction enzyme sites at the 5′ and 3′ ends of the cassetteallow for easy insertion into a pre-existing vector.

Viral Infected Plant Material:

Viruses are a unique class of infectious agents whose distinctivefeatures are their simple organization and their mechanism ofreplication. In fact, a complete viral particle, or virion, may beregarded mainly as a block of genetic material (either DNA or RNA)capable of autonomous replication, surrounded by a protein coat andsometimes by an additional membranous envelope such as in the case ofalpha viruses. The coat protects the virus from the environment andserves as a vehicle for transmission from one host cell to another.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaV, TMV and BV. Transformation of plants usingplant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553(TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809(BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications inMolecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, NewYork, pp. 172-189 (1988). Pseudovirus particles for use in expressingforeign DNA in many hosts, including plants, is described in WO87/06261.

Construction of plant RNA viruses for the introduction and expression ofnon-viral foreign genes in plants is demonstrated by the abovereferences as well as by Dawson, W. O. et al., Virology (1989)172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al.Science (1986) 231:1294-1297; and Takamatsu et al. FEBS Letters (1990)269:73-76.

When the virus is a DNA virus, the constructions can be made to thevirus itself Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by transcribing the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression ofnon-viral foreign genes in plants is demonstrated by the abovereferences as well as in U.S. Pat. No. 5,316,931

In one embodiment, a plant viral nucleic acid is provided in which thenative coat protein coding sequence has been deleted from a viralnucleic acid, a non-native plant viral coat protein coding sequence anda non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral nucleic acid, andensuring a systemic infection of the host by the recombinant plant viralnucleic acid, has been inserted. Alternatively, the coat protein genemay be inactivated by insertion of the non-native nucleic acid sequencewithin it, such that a fusion protein is produced. The recombinant plantviral nucleic acid may contain one or more additional non-nativesubgenomic promoters. Each non-native subgenomic promoter is capable oftranscribing or expressing adjacent genes or nucleic acid sequences inthe plant host and incapable of recombination with each other and withnative subgenomic promoters. Non-native (foreign) nucleic acid sequencesmay be inserted adjacent the native plant viral subgenomic promoter orthe native and a non-native plant viral subgenomic promoters if morethan one nucleic acid sequence is included. The non-native nucleic acidsequences are transcribed or expressed in the host plant under controlof the subgenomic promoter to produce the desired products.

In a second embodiment, a recombinant plant viral nucleic acid isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral nucleic acid isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral nucleic acid. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native nucleic acid sequencesmay be inserted adjacent the non-native subgenomic plant viral promoterssuch that said sequences are transcribed or expressed in the host plantunder control of the subgenomic promoters to produce the desiredproduct.

In a fourth embodiment, a recombinant plant viral nucleic acid isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral nucleic acid to produce a recombinant plantvirus. The recombinant plant viral nucleic acid or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral nucleic acid is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)in the host to produce the desired fusion protein.

Fusion Protein Compartmentalization—Signal Peptides:

As already mentioned hereinabove, compartmentalization of the fusionprotein is an important feature of the present invention because itallows undisturbed plant growth. Thus, according to one aspect of thepresent invention, the fusion protein is compartmentalized within cellsof the plant or cultured plant cells, so as to be sequestered from cellwalls of the cells of the plant or cultured plant cells.

The fusion protein can be compartmentalized within a cellularcompartment, such as, for example, the cytoplasm, endoplasmic reticulum,golgi apparatus, oil bodies, starch bodies, chloroplastids,chloroplasts, chromoplastids, chromoplasts, vacuole, lysosomes,mitochondria or the nucleus.

Accordingly, the heterologous sequence used while implementing theprocess according to this aspect of the present invention includes (i) afirst sequence encoding a cellulose binding peptide; (ii) a secondsequence encoding a recombinant protein, wherein the first and secondsequences are joined together in frame; and (iii) a third sequenceencoding a signal peptide for directing a protein to a cellularcompartment, the third sequence being upstream and in frame with thefirst and second sequences.

The following provides description of signal peptides which can be usedto direct the fusion protein according to the present invention tospecific cell compartments.

It is well-known that signal peptides serve the function oftranslocation of produced protein across the endoplasmic reticulummembrane. Similarly, transmembrane segments halt translocation andprovide anchoring of the protein to the plasma membrane, see, Johnson etal. The Plant Cell (1990) 2:525-532; Sauer et al. EMBO J. (1990)9:3045-3050; Mueckler et al. Science (1985) 229:941-945. Mitochondrial,nuclear, chloroplast, or vacuolar signals target expressed proteincorrectly into the corresponding organelle through the secretorypathway, see, Von Heijne, Eur. J. Biochem. (1983) 133:17-21; Yon Heijne,J. Mol. Biol. (1986) 189:239-242; Iturriaga et al. The Plant Cell (1989)1:381-390; McKnight et al., Nucl. Acid Res. (1990) 18:4939-4943;Matsuoka and Nakamura, Proc. Natl. Acad. Sci. USA (1991) 88:834-838. Arecent book by Cunningham and Porter (Recombinant proteins from plants,Eds. C. Cunningham and A. J. R. Porter, 1998 Humana Press Totowa, N.J.)describe methods for the production of recombinant proteins in plantsand methods for targeting the proteins to different compartments in theplant cell. In particular, two chapters therein (14 and 15) describedifferent methods to introduce targeting sequences that results inaccumulation of recombinant proteins in compartments such as ER,vacuole, plastid, nucleus and cytoplasm. The book by Cunningham andPorter is incorporated herein by reference. Presently, the preferredsite of accumulation of the fusion protein according to the presentinvention is the ER using signal peptide such as Cel 1 or the riceamylase signal peptide at the N-terminus and an ER retaining peptide(HDEL or KDEL) at the C-terminus.

Promoters and Control of Expression:

Any promoter which can direct the expression of the fusion proteinaccording to the present invention can be utilized to implement theprocess of the instant invention, both constitutive and tissue specificpromoters. According to presently preferred embodiment the promoterselected is constitutive, because such a promoter can direct theexpression of higher levels of the fusion protein. In this respect thepresent invention offers a major advantage over the teachings of U.S.Pat. No. 5,474,925 in which only tissue specific and weak promoters canbe employed because of the deleterious effect of the fusion proteindescribed therein on cell wall development. The reason for which thepresent invention can utilize strong and constitutive promoters reliesin the compartmentalization and sequestering approach which prohibitscontact between the expressed fusion protein and the plant cell wallswhich such walls are developing.

Constitutive and tissue specific promoters, CaMV35S promoter (Odell etal. Nature (1985) 313:810-812) and ubiquitin promoter (Christensen andQuail, Transgenic research (1996) 5:213-218) are the most commonly usedconstitutive promoters in plant transformations and are the preferredpromoters of choice while implementing the present invention.

In corn, within the kernel, proteins under the ubiquitin promoters, arepreferentially accumulated in the germ (Kusnadi et al., Biotechnol.Bioeng. (1998) 60:44-52). The amylose-extender (Ae) gene encodingstarch-branching enzyme IIb (SBEIIb) in maize is predominantly expressedin endosperm and embryos during kernel development (Kim et al. Plant.Mol. Biol. (1998) 38:945-956). A starch branching enzyme (SBE) showedpromoter activity after it was introduced into maize endospermsuspension cells by particle bombardment (Kim et al. Gene (1998)216:233-243). In transgenic wheat it has been shown that a native HMW-GSgene promoter can be used to obtain high levels of expression of seedstorage and, potentially, other proteins in the endosperm (Blechl andAnderson, Nat. Biotechnol. (1996) 14:875-9). Polygalacturonase (PG)promoter was shown to confer high levels of ripening-specific geneexpression in tomato (Nicholass et al. Plant. Mol. Biol. (1995)28:423-435). The ACC oxidase promoter (Blume and Grierson, Plant. J.(1997) 12:731-746) represents a promoter from the ethylene pathway andshows increased expression during fruit ripening and senescence intomato. The promoter for tomato 3-hydroxy-3-methylglutaryl coenzyme Areductase gene accumulates to high level during fruit ripening(Daraselia et al. Plant. Physiol. (1996) 112:727-733). Specific proteinexpression in potato tubers can be mediated by the patatin promoter(Sweetlove et al. Biochem. J. (1996) 320:487-492). Protein linked to achloroplast transit peptide changed the protein content in transgenicsoybean and canola seeds when expressed from a seed-specific promoter(Falco et al. Biotechnology (NY) (1995) 13:577-82). The seed specificbean phaseolin and soybean beta-conglycinin promoters are also suitablefor the latter example (Keeler et al. Plant. Mol. Biol. (1997)34:15-29). Promoters that are expressed in plastids are also suitable inconjunction with plastid transformation.

Each of these promoters can be used to implement the process accordingto the present invention.

Thus, the plant promoter employed can a constitutive promoter, a tissuespecific promoter, an inducible promoter or a chimeric promoter.

Examples of constitutive plant promoters include, without being limitedto, CaMV35S and CaMV19S promoters, FMV34S promoter, sugarcanebacilliform badnavirus promoter, CsVMV promoter, Arabidopsis ACT2/ACT8actin promoter, Arabidopsis ubiquitin UBQ1 promoter, barley leaf thioninBTH6 promoter, and rice actin promoter.

Examples of tissue specific promoters include, without being limited to,bean phaseolin storage protein promoter, DLEC promoter, PHSβ promoter,zein storage protein promoter, conglutin gamma promoter from soybean,AT2S1 gene promoter, ACT11 actin promoter from Arabidopsis, napApromoter from Brassica napus and potato patatin gene promoter.

The inducible promoter is a promoter induced by a specific stimuli suchas stress conditions comprising, for example, light, temperature,chemicals, drought, high salinity, osmotic shock, oxidant conditions orin case of pathogenicity and include, without being limited to, thelight-inducible promoter derived from the pea rbcS gene, the promoterfrom the alfalfa rbcS gene, the promoters DRE, MYC and MYB active indrought; the promoters INT, INPS, prxEa, Ha hsp17.7G4 and RD21 active inhigh salinity and osmotic stress, and the promoters hsr303J and str246Cactive in pathogenic stress.

Expression Follow Up:

Expression of the fusion protein can be monitored by a variety ofmethods. For example, ELISA or western blot analysis using antibodiesspecifically recognizing the recombinant protein or its cellulosebinding peptide counterpart can be employed to qualitatively and/orquantitatively monitor the expression of the fusion protein in theplant. Alternatively, the fusion protein can be monitored by SDS-PAGEanalysis using different staining techniques, such as, but not limitedto, coomasie blue or silver staining. Other methods can be used tomonitor the expression level of the RNA encoding for the fusion protein.Such methods include RNA hybridization methods, e.g., Northern blots andRNA dot blots.

Thus, according to the present invention there is provided a geneticallymodified or viral infected plant or cultured plant cells expressing afusion protein including an enzyme being capable of catalyzing theoxidation of phenolic groups and a cellulose binding peptide.

According to a preferred embodiment of the present invention the fusionprotein is compartmentalized within cells of said plant or culturedplant cells, so as to be sequestered from cell walls of said cells ofsaid plant or cultured plant cells, so as not to hamper development andto allow higher expression, if so required. According to a preferredembodiment the fusion protein is compartmentalized within a cellularcompartment selected from the group consisting of cytoplasm, endoplasmicreticulum, golgi apparatus, oil bodies, starch bodies, chloroplastids,chloroplasts, chromoplastids, chromoplasts, vacuole, lysosomes,mitochondria, and nucleus.

Determination of Oxidase and Peroxidase Activity:

When employing a polynucleotide encoding a laccase in the process of theinvention, an amount of laccase in the range of 0.02-2000 laccase units(LACU) per gram of dry lignocellulosic material will generally besuitable; when employing peroxidases, an amount thereof in the range of0.02-2000 peroxidase units (PODU) per gram of dry lignocellulosicmaterial will generally be suitable.

The determination of oxidase (e.g., laccase) activity is based on theoxidation of syringaldazin to tetramethoxy azo bis-methylene quinoneunder aerobic conditions, and 1 LACU is the amount of enzyme whichconverts 1 μM of syringaldazin per minute under the followingconditions: 19 μM syringaldazin, 23.2 mM acetate buffer, 30° C., pH 5.5,reaction time 1 minute, shaking; the reaction is monitoredspectrophotometrically at 530 nm.

With respect to peroxidase activity, 1 PODU is the amount of enzymewhich catalyses the conversion of 1 μmol of hydrogen peroxide per minuteunder the following conditions: 0.88 mM hydrogen peroxide, 1.67 mM2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate), 0.1 M phosphatebuffer, pH 7.0, incubation at 30° C.; the reaction is monitoredphotometrically at 418 nm.

Binding of the Fusion Protein to the Plant Derived Cellulosic Matter:

When sufficient expression has been detected, binding of the fusionprotein to the plant derived cellulosic matter is effected. Such bindingcan be achieved, for example, as follows. Whole plants, plant derivedtissue or cultured plant cells are homogenized by mechanical method inthe presence or absence of a buffer, such as, but not limited to, PBS.The fusion protein is therefore given the opportunity to bind to theplant derived cellulosic matter. Buffers that may include salts and/ordetergents at optimal concentrations may be used to wash non specificproteins from the cellulosic matter.

Thus, further according to the present invention there is provided acomposition of matter comprising a cell wall preparation derived from agenetically modified or virus infected plant or cultured plant cellsexpressing a fusion protein including an enzyme being capable ofcatalyzing the oxidation of phenolic groups and a cellulose bindingpeptide, said fusion protein being immobilized to cellulose in said cellwall preparation via said cellulose binding peptide.

Oxidizing Agents:

The enzyme(s) and oxidizing agent(s) used in the process of theinvention should clearly be matched to one another, and it is clearlypreferable that the oxidizing agent(s) in question participate(s) onlyin the oxidative reaction involved in the binding process, and does/donot otherwise exert any deleterious effect on the substances/materialsinvolved in the process.

Oxidases, e.g. laccases, are, among other reasons, well suited in thecontext of the invention since they catalyze oxidation by molecularoxygen. Thus, reactions taking place in vessels open to the atmosphereand involving an oxidase as enzyme will be able to utilize atmosphericoxygen as oxidant; it may, however, be desirable to forcibly aerate thereaction medium during the reaction to ensure an adequate supply ofoxygen.

In the case of peroxidases, hydrogen peroxide is a preferred peroxide inthe context of the invention and is suitably employed in a concentration(in the reaction medium) in the range of 0.01-100 mM.

pH in the Reaction Medium:

Depending, inter alia, on the characteristics of the enzyme(s) employed,the pH in the aqueous medium (reaction medium) in which the process ofthe invention takes place will be in the range of 3-10, preferably inthe range 4-9.

General Procedures:

Generally, the nomenclature used herein and the laboratory proceduresutilized when practicing the present invention include molecular,biochemical, microbiological and recombinant DNA techniques. Suchtechniques are thoroughly explained in the literature. See, for example,“Molecular Cloning: A laboratory Manual” Sambrook et al., (1989);“Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M.,ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”,John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Product by Process:

The present invention also relates to a lignocellulose-based productobtainable by a process according to the invention as disclosed herein.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications cited herein are incorporatedby reference in their entirety.

1. A process for the manufacture of a lignocellulose product, theprocess comprising the step of mixing in a reaction medium: (i) aphenolic polymer being substituted with a phenolic hydroxy group; (ii) alignocellulose containing material having immobilized to a cellulosicfraction thereof a fusion polypeptide, said fusion polypeptide includingan enzyme being capable of catalyzing the oxidation of phenolic groupsand a cellulose binding peptide; and (iii) an oxidizing agent.
 2. Theprocess of claim 1, wherein said lignocellulose product is selected fromthe group consisting of fiber board, particle board, flakeboard, plywoodand molded composites.
 3. The process of claim 1, wherein saidlignocellulose product is selected from the group consisting of paperand paperboard.
 4. The process of claim 1, wherein said lignocellulosecontaining material is a cell wall preparation derived from agenetically modified or virus infected plant or cultured plant cellsexpressing said fusion protein.
 5. The process of claim 1, wherein saidlignocellulose containing material is selected from the group consistingof vegetable fiber and wood fiber derived from a genetically modified orvirus infected plant expressing said fusion polypeptide.
 6. The processof claim 1, wherein the phenolic substituent is selected from the groupconsisting of p-coumaric acid, p-coumaryl alcohol, coniferyl alcohol,sinapyl alcohol, ferulic acid and p-hydroxybenzoic acid.
 7. The processof claim 1, wherein said phenolic polymer forms an integral part of saidlignocellulose containing material.
 8. The process of claim 7, whereinsaid phenolic polymer is lignin.
 9. The process of claim 1, wherein saidphenolic polymer is a phenolic polysaccharide.
 10. The process of claim9, wherein the polysaccharide portion of the phenolic polysaccharide isselected from the group consisting of modified and unmodified starches,modified and unmodified cellulose, and modified and unmodifiedhemicelluloses.
 11. The process of claim 9, wherein said phenolicpolysaccharide is selected from the group consisting of ferulylatedarabinoxylans and ferulylated pectins.
 12. The process of claim 1,wherein said reaction medium is incubated for a period of from 1 minuteto 10 hours.
 13. The process of claim 12, wherein said fusionpolypeptide is incubated in the presence of said oxidizing agent for aperiod of from 1 minute to 10 hours.
 14. The process of claim 1, whereinsaid enzyme is selected from the group consisting of oxidases andperoxidases.
 15. The process of claim 1, wherein said enzyme is anoxidase selected from the group consisting of laccases (EC 1.10.3.2),catechol oxidases (EC 1.10.3.1) and bilirubin oxidases (EC 1.3.3.5), andsaid oxidizing agent is oxygen.
 16. The process of claim 15, whereinsaid enzyme is a laccase and is present in an amount in the range of0.02-2000 LACU per g of dry lignocellulose.
 17. The process of claim 15,wherein said reaction medium is aerated.
 18. The process of claim 15,wherein said enzyme is a laccase encoded by a polynucleotide obtainedfrom a fungus of the genus Botrytis, Myceliophthora, Trametes or theplant Acer pseudoplanus.
 19. The process of claim 18, wherein the fungusis Trametes versicolor or Trametes villosa.
 20. The process of claim 1,wherein said enzyme is a peroxidase and said oxidizing agent is hydrogenperoxide.
 21. The process of claim 20, wherein said peroxidase ispresent in an amount in the range of 0.02-2000 PODU per g of drylignocellulose, and the initial concentration of hydrogen peroxide inthe reaction medium is in the range of 0.01-100 mM.
 22. The process ofclaim 1, wherein the amount of lignocellulose employed corresponds to0.1-90% by weight of the reaction medium, calculated as drylignocellulose.
 23. The process of claim 1, wherein the temperature ofthe reaction medium is in the range of 10°-120° C.
 24. The process ofclaim 23, wherein the temperature of the reaction medium is in the rangeof 15°-90° C.
 25. The process of claim 1, wherein an amount of saidphenolic polymer in the range of 0.1%-10% by weight.
 26. The process ofclaim 1, wherein the pH in the reaction medium is in the range of 3-10.27. The process of claim 26, wherein the pH in the reaction medium is inthe range of 4-9.
 28. The process of claim 1, wherein the reactionmedium further comprising a lignocellulose containing material devoid ofsaid fusion protein.
 29. The process of claim 28, wherein saidlignocellulose containing material devoid of said fusion protein isselected from the group consisting of vegetable fiber, wood fiber, woodchips, wood flakes, wood veneer and recycled fibers.
 30. Alignocellulose product obtainable by the process of claim
 1. 31. Agenetically modified or viral infected plant or cultured plant cellsexpressing a fusion protein including an enzyme being capable ofcatalyzing the oxidation of phenolic groups and a cellulose bindingpeptide.
 32. The genetically modified or viral infected plant orcultured plant cells of claim 31, wherein said fusion protein beingcompartmentalized within cells of said plant or cultured plant cells, soas to be sequestered from cell walls of said cells of said plant orcultured plant cells.
 33. The genetically modified or viral infectedplant or cultured plant cells of claim 31, wherein expression of saidfusion protein is under a control of a constitutive or tissue specificplant promoter.
 34. The genetically modified or viral infected plant orcultured plant cells of claim 32, wherein said fusion protein iscompartmentalized within a cellular compartment selected from the groupconsisting of cytoplasm, endoplasmic reticulum, golgi apparatus, oilbodies, starch bodies, chloroplastids, chloroplasts, chromoplastids,chromoplasts, vacuole, lysosomes, mitochondria, and nucleus.
 35. Acomposition of matter comprising a cell wall preparation derived from agenetically modified or virus infected plant or cultured plant cellsexpressing a fusion protein including an enzyme being capable ofcatalyzing the oxidation of phenolic groups and a cellulose bindingpeptide, said fusion protein being immobilized to cellulose in said cellwall preparation via said cellulose binding peptide.
 36. A nucleic acidmolecule comprising: (a) a promoter sequence for directing proteinexpression in plant cells; and (b) a heterologous nucleic acid sequenceincluding: (i) a first sequence encoding a cellulose binding peptide;(ii) a second sequence encoding an enzyme being capable of catalyzingthe oxidation of phenolic groups, wherein said first and secondsequences are joined together in frame.
 37. The nucleic acid molecule ofclaim 36, further comprising a sequence element selected from the groupconsisting of an origin of replication for propagation in bacterialcells, at least one sequence element for integration into a plant'sgenome, a polyadenylation recognition sequence, a transcriptiontermination signal, a sequence encoding a translation start site, asequence encoding a translation stop site, plant RNA virus derivedsequences, plant DNA virus derived sequences, tumor inducing (Ti)plasmid derived sequences, a transposable element derived sequence and aplant operative signal peptide for directing a protein to a cellularcompartment of a plant cell.
 38. The nucleic acid molecule of claim 36,wherein said cellular compartment is selected from the group consistingof cytoplasm, endoplasmic reticulum, golgi apparatus, oil bodies, starchbodies, chloroplastids, chloroplasts, chromoplastids, chromoplasts,vacuole, lysosomes, mitochondria, and nucleus.